Prehydrolysis of lignocellulose

ABSTRACT

The invention relates to the prehydrolysis of lignocellulose by passing an acidic or alkaline solution through solid lignocellulosic particles with removal of soluble components as they are formed. The technique permits a less severe combination of pH, temperature and time than conventional prehydrolysis. Furthermore, greater extraction of both hemicellulose and lignin occurs simultaneously in the same reactor and under the same conditions.

The United States Government has rights in this invention under ContractNo. DE ACO2-83CH10093 between the United States Department of Energy andthe National Renewable Energy Laboratory, a division of the MidwestResearch Institute.

This is a Division of application Ser. No. 08/126,792 filed Sep. 24,1993.

FIELD OF THE INVENTION

The invention relates to certain improvements in the prehydrolysis oflignocellulosic feedstocks to fractionate it into hemicellulose,cellulose and lignin. The carbohydrate fractions may then be hydrolysedinto sugars and fermented to produce alcohol and other products.

BACKGROUND TO THE INVENTION

Lignocellulose is ubiquitous in all wood species and all agriculturaland forestry waste. In addition, municipal waste which typicallycontains about half waste paper and yard waste, is a source oflignocellulosic materials. Currently, municipal waste is buried orburned at considerable expense to the disposer or the governmentorganization providing solid waste services.

Lignocellulosic biomass is a complex structure of cellulose fiberswrapped in a lignin and hemicellulose sheath. The ratio of the threecomponents varies depending on the type of biomass. Typical ratios areas follows:

    ______________________________________              Softwoods Corn cobs R D F*    ______________________________________    Cellulose   42%         40%       52%    Hemicellulose                25%         36%       26%    Lignin      28%         13%       20%    ______________________________________     *RDF = Refuse Derived Fuel from municipal systems waste

Different woods also have different compositions. Softwoods(gymnosperms) generally have more glucommanans and less glucuronoxylansthan hardwoods and grasses (angiosperms).

Cellulose is a polymer of D-glucose with β 1→4! linkages between each ofthe about 500 to 10,000 glucose units. Hemicellulose is a polymer ofsugars, primarily D-xylose with other pentoses and some hexoses with β1→4! linkages. Lignin is a complex random polyphenolic polymer.Therefore, lignocellulose represents a very cheap and readily availablesubstrate for the preparation of sugars which may be used alone ormicrobially fermented to produce alcohols and other industrialchemicals.

Ethanol, one of the alcohols which can be produced from lignocellulosicbiomass, has a number of industrial and fuel uses. Of particularinterest is the use of ethanol as an additive to gasoline to boostoctane, reduce pollution and to partially replace gasoline in themixture. This composition is the well known commercial product called"gasohol". It has been proposed to eliminate gasoline completely fromthe fuel and to burn ethanol alone. Such a fuel would produceconsiderably less air pollution by not forming as much carbon monoxideor hydrocarbon emissions. Furthermore, gasoline is produced from crudeoil which fluctuates in price, availability and is the subject ofunpredictable world politics.

It has been estimated that about 1×10⁹ tons of lignocellulosic wastesare produced every year. This amount exceeds the total amount of crudeoil consumed per year. In theory, if properly managed, thelignocellulose produced by the United States is sufficient to produceall of the country's needs for liquid fuel if the cellulose andhemicellulose can be completely converted into ethanol. The amount ofenergy theoretically obtainable from the combustion of cellulose or theglucose or alcohol derived therefrom is about 7200 BTU per pound orroughly equivalent to 0.35 pounds of gasoline. Hemicellulose has similarvalue when converted into sugars or ethanol. Consequently, cellulose andhemicellulose represents a readily available potential source forethanol production.

The technology for the production of ethanol from grain and fruit forbeverage purposes has been well developed for centuries. However, thecosts have been relatively high compared to the cost of gasoline.Accordingly, many methods have been proposed to reduce the cost andincrease the efficiency of ethanol production.

Among the techniques proposed for the production of fuel grade ethanolinclude the hydrolysis of cellulose to produce glucose which can befermented to produce ethanol. Cellulose in the form of wood, newsprintand other paper, forest, agricultural, industrial and municipal wastesis quite inexpensive compared to grain, fruit, potatoes or sugarcanewhich is traditionally used to prepare alcohol beverages.

Cellulose hydrolysis using an acid catalyst to produce sugars has beenknown for decades but can be costly and requires special equipment. Thesugars themselves, are somewhat labile to the harsh hydrolysisconditions and a large number of unwanted or toxic byproducts may beformed. If exposed to acid for too long, the glucose derived fromcellulose degrades into hydroxymethylfurfural which can be furtherdegraded into levulinic acid and formic acid. Xylose, which is formedfrom hemicellulose, is degraded by acids into furfural and then resultsin tars and other degradation products.

In order for acid to completely hydrolyse the cellulose andhemicellulose in a lignocellulosic substrate, degradation of thedesirable sugars and formation of the toxic byproducts cannot be avoideddue to kinetic constraints. To use conditions sufficiently gentle thatinsignificant degradation of sugars will occur does not result incomplete hydrolysis of substrate. Furthermore, the acid is corrosive andrequires special handling and equipment. Accordingly, in the last twentyyears attention has focused on enzymatic hydrolysis of cellulose withcellulase followed by fermentation of the resulting sugars to produceethanol which in turn is distilled to purify it sufficiently for fueluses.

Cellulase is an enzyme complex that includes three different types ofenzymes involved in the saccharification of cellulose. The cellulaseenzyme complex produced by Trichoderma reesei QM 9414 contains theenzymes named endoglucanase (E.C.3.2.1.4), cellobiohydrolase(E.C.3.2.1.91) and β-glucosidase (E.C.3.2.1.21). Gum et al, Biochem.Biophys. Acta, 446: 370-86 (1976). The combined synergistic actions ofthese three enzymes in the cellulase preparation completely hydrolysescellulose to D-glucose.

However, cellulase can not completely degrade the cellulose found innative, unpretreated lignocellulose. It appears that the hemicelluloseand lignin interfere with the access of the enzyme complex to thecellulose, probably due to their coating of the cellulose fibers.Furthermore, lignin itself can bind cellulase thereby rendering itinactive or less effective for digesting cellulose. For example, rawground hardwood is only about 10 to 20% digestible into sugars using acellulase preparation.

To overcome these shortcomings, applicants and others have previouslydisclosed a pretreatment step which attempts to degrade or remove atleast a portion of the hemicellulose and/or lignin. The result of thepretreatment has been greater digestibility of the cellulose by acellulase complex. One such pretreatment has been the use of acomminution step and a combination of heat and acid for a period of timewhich hydrolyses most of the hemicellulose, thus rendering the cellulosedigestible by a cellulase complex.

In the prior art, by using such a pretreatment step, little lignin hasbeen removed. See Grohmann et al, Biotechnology and Bioengineering,Symp. No. 17: 135-151 (1986), Torget et al, Applied Biochemistry andBiotechnology, 24/25: 115-126 (1990), Torget et al, Applied Biochemistryand Biotechnology, 28/29: 75-86 (1991) and Torget et al, AppliedBiochemistry and Biotechnology, 34/35: 115-123 (1992).

Lignin removal from cellulosic fibers by a caustic (alkali) is the basisfor Kraft pulping and paper making. However, such techniques are aimedat conserving the polymeric carbohydrate integrity and thus do notproduce simple sugars, and do not separate the hemicellulose from thecellulose.

The difficulty of degrading the hemicellulose and cellulose remains.Conditions optimized to remove hemicellulose are not very effective atremoving lignin and vice versa. Therefore, the cellulose remaining afterprehydrolysis may be less than ideally separated from the otherconstituents and may retard digestion by cellulase. Furthermore, thecosts involved for the pretreatment step can be significant. Thechemicals used to alter pH and the steam required to heat thelignocellulose add a cost to the process. The greater the duration ofthe prehydrolysis step, the greater cost in heat to maintain thetemperature; and the slower the overall process, also the greater costin time and equipment.

Elian et al, U.S. Pat. No. 2,734,836, discloses a process where acid isused to pretreat cellulosic materials to extract pentoses using aceticacid. The material is sprinkled with the acid and heated to 80°-120° C.and the acid is recycled through the cooker in a manner to preserve thecellulose fibers. The residual material is used in conventional pulping.

Richter, U.S. Pat. No. 3,532,594, discloses digesting cellulosicmaterial by soaking the solids in an alkaline liquid, and then applyingsteam in a gas phase to heat the material. The material is cooled andwashed to recover cellulosic fibers. The digestion of the non-celluloseoccurs in the gas phase as wood chips descend in the reactor. Noreaction is occurring in the countercurrent washing step.

Eickemeyer, U.S. Pat. No. 3,787,241, discloses a percolator vessel fordecomposing portions of wood. The first stage is the hydrolysis ofhemicellulose to xylose using 1% sulfuric acid (column 4, lines 23-34)and then acid hydrolysis of cellulose occurs later. Lignin remains inthe reactor throughout the hydrolysis and is removed at the end.

Pfeiffer, U.S. Pat. No. 4,226,638, discloses an acid pretreatment ofyoung plants where the acid is not permitted to saturate the youngplants. Column 3, line 59 specifically states that the amount ofsaturation is less than 70% and line 67 states that 60% of the xylan ishydrolysed. The wash step is performed by water. The goal is to extractxylose form plant material.

Brink, U.S. Pat. No. 4,384,897 and later U.S. Pat. No. 5,221,357, uses anitric acid two stage hydrolysis where the first stage is under mildconditions to hydrolyse the hemicellulose. (pH 2-3, 140°-220° C.) Laterharsher acidic conditions are used to hydrolyse the cellulose presentwhich is then washed free from the lignin. No lignin is removed duringthe hydrolysis. The liquid and biomass particles travel in a cocurrentflow pattern according to column 10 lines 1-5 of the more recent patent.

Elmore, U.S. Pat. No. 4,436,586, describes a method for treating woodchips by acid prehydrolysis to extract xylose for fermentation toethanol and preserves the cellulose fibers and lignin for kraft pulpingof the residual solids. The pretreatment is in a reactor with filteringscreens and recycling of the acid.

Tourier et al, U.S. Pat. No. 4,511,433, describes a continuousextraction process where wood is placed in a reactor 1 and is retainedby filter 2 where a strongly acidic solution is passed through thesystem to remove pentoses. The acidic solution is mixed with a phenolcompound in a different phase to remove lignin. The two phase system isneeded for Tourier to achieve their results.

Neves, U.S. Pat. No. 4,564,595, discloses a process which includescellulose cooking and degradation with acid. The process separateshemicellulose from the pretreated material and in a separate stepremoves lignin. According to the second paragraph in column 7, agitationof the mixture appears essential to adequate extraction.

Sherman et al, U.S. Pat. No. 4,612,286 and later U.S. Pat. No.4,668,340, hydrolyse the hemicellulose in a high solids system by acomplex process of passing a 20-40% solids slurry of biomass into thebottom of a reactor and passing acid through a series of screens andsolid-liquid separators to wash the hydrolysed C5 sugars in a separatelocation which are then withdrawn as hydrolyzate. According to column 2,line 37, the acid concentration in the reaction chamber is about 2% to10%. Conditions are chosen so that cellulose is not hydrolysed. Theremaining solids include cellulose and lignin which are burned or ifseparated, must be done in a separate step.

Wright, U.S. Pat. No. 4,615,742, discloses a series of hydrolysisreactors. Some of these are prehydrolysis reactors and are for removinghemicellulose while others are for hydrolysis. Because the contents movein a series, the duration of each step is the same. The process does notremove lignin from the solids and multiple reactors are required. Withineach reactor is a predefined set of conditions.

Rehberg, U.S. Pat. No. 4,995,943, pretreats biomass with high pressurecarbon dioxide and then suddenly releases it to open up the material toallow better degradation of cellulose. The gas applied is described asanhydrous and therefore should not contain carbonic acid other than thatwhich forms in situ. The purpose of the Rehberg process is unrelated toany prehydrolysis process as water is not present in appreciableamounts.

Grohmann et al, U.S. Pat. No. 5,125,977, demonstrated that differentxylans could be removed during prehydrolysis under two differentconditions by prehydrolyzing the substrate, centrifuging the mixture torecover xylose from certain xylans in the supernatant. The solids wereremixed with additional acid, the prehydrolysis completed and a secondxylose solution produced by apparently different xylans in thehemicellulose. The two types of xylan are hydrolysed by two differentconditions which are optimized for each xylan.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the previousdifficulties in prehydrolyzing lignocellulosic material.

It is another object of the present invention to use a less severecombination of conditions to effect prehydrolysis of lignocellulose.

It is a further object of the present invention to separate a greaterpercentage of hemicellulose and lignin from cellulose in alignocellulosic substrate.

It is yet another object of the present invention to prepare acellulosic material which is more easily digested by a given amount ofcellulase and a hydrolyzate liquid with a greater proportion of thehemicellulose and lignin.

It is still another object of the present invention to enzymaticallyproduce sugars from pretreated lignocellulosic substrates.

It is still a further object of the present invention to produce aliquid and solid stream containing carbohydrates from a lignocellulosicfeedstock which are amenable to fermentation processes to producealcohol and other industrial chemicals.

It is an additional object of the present invention to separate liquidproducts produced by prehydrolysis from a solid residue during theprehydrolysis before the conditions are returned to ambient conditions.

It is yet another object of the present invention to vary the conditionsof time, temperature and acid concentration in a single reactor during asingle prehydrolysis step by using the concept of a varying combinedseverity factor so that the resultant solid residue is amenable toenzymatic saccharification as well as acid catalyzed saccharification.

It is yet an additional object of the present invention to adjust the pHbefore enzymatic saccharification of cellulose in such a way that theproduct of neutralization, i.e. gypsum, is less soluble and thus removedto a greater extent.

The present invention utilizes a flow-through reactor such as apercolation reactor containing lignocellulosic substrate whereprehydrolysis fluid is passed through the lignocellulosic substrate andremoved while still hot. The fluid may be further treated to completelyhydrolyze any sugar polymers or oligomers. The solid residue from theprehydrolysis reactor, after being washed with reaction temperaturewater in the reactor, is then contacted with cellulase to saccharify thecellulose into sugars. Both sugar solutions may be fermented separately,together and/or simultaneously to produce industrial chemicals such asethanol with the cellulase saccharifying the cellulose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the entire process.

FIG. 2 is a schematic view of exemplary apparatus used in the Examples.

FIG. 3a is the result of HPLC separation of components of theprehydrolyzate.

FIG. 3b is the result of HPLC separation of components of theprehydrolyzate after it has been further acid hydrolysed.

FIG. 4a displays the percentage of cellulose digested by cellulase forthe pretreated product of the present invention as circles and forα-cellulose control as triangles.

FIG. 4b displays the percentage of ethanol which can be producedtheoretically from fermentation of the given amount of cellulose in asimultaneous saccharification and fermentation process. The pretreatedproduct of the present invention is shown as circles and α-cellulosecontrol is shown as triangles.

FIG. 5 displays the percentage of cellulose digested over time for hotwater prehydrolysis. In the legend, the first number represents time andthe second represents temperature employed during the prehydrolysis.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Prehydrolysis of lignocellulose involves at least the partial separationof hemicellulose and/or lignin from cellulose so that a more purifiedcellulose is produced. Prehydrolysis is generally performed by theaction of chemicals, particularly chemicals which change the pH, heatand water. Depending on the conditions, different components areseparated at different rates. Furthermore, depending on the compositionand nature of the particular lignocellulosic substrate, differentcombinations of conditions will be needed.

In the present invention, the different components liberated from thelignocellulosic solids may be continuously removed under a single set ofconditions, plural sets of conditions or under constantly changingconditions. This is performed by a process and apparatus novel to thefield of lignocellulose prehydrolysis.

While applicants do not wish to be bound by any particular theory, theybelieve that the solubilized lignin and breakdown products are moreeasily separated from the solid lignocellulosic substrates when thecomposition is hot, i.e. at the temperature used during prehydrolysis.When the composition is allowed to cool after heat and chemicalprehydrolysis treatment and before washing the solid material, lesshemicellulose and lignin are removed. It appears that the degraded orpartially degraded hemicellulose and lignin precipitate or recondenseson the lignocellulosic substrate as it cools and is not easily washedaway. Regardless of any particular theory, less separation is noticed.

During the partial removal of hemicellulose and lignin, it is believedthat "pores" or the like are created in the hemicellulose/lignin coatingwhich, in later steps, allows cellulase to enter and digest thecellulose. As more lignin and hemicellulose are removed, the "pores" arelarger and allow greater contact between cellulose and cellulase. Thisis believed to be the cause of the greater digestibility noticed in thepresent invention.

The present invention utilizes a flow-through system where fluid moveswith respect to the solid lignocellulose. The lignocellulose solids maybe stationary, travel in a counter-current or cross-current fashion. Itis even possible for the system to use a co-current or stationary systemwhich is agitated. One typical design is a percolation reactor. One canperform a solid-liquid separation in the flow-through system by using ascrew-like device to cause the separation continuously during or at theend of prehydrolysis. Important to the process is the movement andremoval of fluid during the prehydrolysis to separate soluble productsas they are released from the solid lignocellulosic residue.

Fluid need not be flowing constantly, but may be pulsed or stopped for aperiod of time, but it does need to move at least part of the timebefore the end of the prehydrolysis process. Alternatively, a pulsedsystem may blow air or other inert gas through the system to help pushout the prehydrolyzate. An air pulse may also provide an overpressure orsimply to agitate the system.

A continuous prehydrolysis reactor may also be used. Such a reactorwould have lignocellulosic material driven through the reactor whilefluid is passed through the material, typically in a counter-current orcross-current manner. For example, if the prehydrolysis reactor is inthe configuration of a column, the lignocellulosic material may beaugured into the bottom of the column and removed from the top whilefluid containing the degrading compound(s) is added at the top andpasses through the biomass to be removed at the bottom. The reverseconfiguration is also possible. Alternatively, the lignocellulosicsubstrate may be driven laterally while fluid is applied on top andallowed to percolate down to be removed at the bottom.

One advantage of the flow-through design is that a much higherpercentage of xylose can be recovered from the prehydrolyzate. Thissystem can recover over 90% of the xylose, galactose, manose andarabinose whereas previous prehydrolysis systems with one set ofconditions recover only 60-80% of the hemicellulosic sugars.Furthermore, radically different amounts of lignin are removed. Previousacid prehydrolysis removes about 5% and at most about 15% of the lignin.By comparison, the acid prehydrolysis of present invention removes atleast 20% of the lignin.

The duration of the prehydrolysis process is defined as the period oftime when the lignocellulosic material is exposed to a predefinedtemperature or temperatures and chemical concentration orconcentrations. While passing fluid by the solid lignocellulosicmaterial throughout the prehydrolysis period is preferred, one couldpass fluid through the reactor only once immediately before the end ofthe prehydrolysis step. Such a variation is exemplified below.

It is recognized that certain components are easy to hydrolyse whereasothers are hard to hydrolyse. Degradation of the soluble products beforetheir removal is not necessary nor particularly desired. Plural orcontinuous passages of fluid may be preferable, depending on thecomposition of the lignocellulosic substrate, for the removal of variouscomponents as they are solubilized.

It is also recognized that at different times during the prehydrolysis,the composition of the resulting liquor may vary. This is particularlylikely if different physical and chemical conditions are used during theprehydrolysis step. Accordingly, different portions of theprehydrolyzate liquor stream need not be mixed together, but rather theliquor stream at one time may be diverted to a different additionaltreatment from another liquor stream. For example, if xylose monomers,xylose oligomers, hexose and lignin predominate in different streams,one may wish to fractionate the prehydrolyzate and further treat thexylose oligomers outside the prehydrolysis reactor to hydrolyse them.Further, different sugars may be sent to different fermentations asdifferent microorganisms can utilize or prefer different sugars.Likewise, lignin predominant fractions may be discarded, subjected tofurther treatment or dewatered and burned.

In the simplest situation, a reactor contains a fluid inlet at one endand a fluid outlet at the opposite end. Lignocellulosic material isadded to the reactor and fluid is passed through it. One or both of thefluid connections may be covered by a screen, frit or other means forretaining lignocellulosic substrate in the reactor. The lignocellulosicmaterial may be prehydrolyzed in a batch mode, semicontinuous or fullycontinuous manner. The solids entrance and/or exit (which may be thesame) are usually different from the fluid inlet and outlet.

Numerous variations on the basic design are possible, the importantfeature being the passage of fluid through or across the lignocellulosicmaterial during prehydrolysis. For example, solid lignocellulosicmaterial may be continuously added and continuously withdrawn from thesystem. Alternatively the solids and liquids may pass in a countercurrent fashion by having the liquids being pumped through or by amoving solid material. The action of gravity or floatation may alsoeffect movement of either the solid or liquid. Artificial means such asa screw, moving screens or pumped air bubbles may also be used to effectmovement. The orientation of the reactor and direction of fluid flow isnot critical and gravity may assist in moving either the fluid or thesolid lignocellulosic material.

Types of flow-through reactors include percolation reactors, screw-typereactors, gravity flow tower reactors, spray and draining reactors,washing reactors and a number of other variations of solid/liquidcontacting and separation systems. The term "flow-through reactor" isintended to cover all of these variations as they all retain solids in azone while liquids flow out of the zone containing the solids andthereby carry hydrolyzed compounds.

The lignocellulosic material is preferably ground before being placed inthe reactor. If the nature of the lignocellulosic material is such thatit will brake down when mixed before of inside the prehydrolysisreactor, then grinding is not necessary. The particle size is notcritical but generally, the smaller the particle size, the faster theprehydrolysis will occur. Smaller particles also can form a tightlypacked bed which will allow less channeling of fluid flow through thebed. Further, smaller particle sizes inherently provide more surfacearea for cellulase to attack and degrade cellulose after prehydrolysis.On the other hand, particles which are too small may form a dense matwhich is difficult for fluid to flow through at an acceptable rate. Verysmall particles are also difficult to retain in the reactor, may requirethe use of a fine screen or frit with its increased cost and reducedflow rates and very small particles may tend to clog the pores of anyretaining means.

Appropriate particle sizes vary with the feedstock and its inherentphysical properties. Particle sizes appropriate for ground wood are inthe range of about 0.1 mm to 30 mm preferably in the range of 0.5 mm to4 mm. Other materials may be larger or smaller depending on theparticular materials, particularly those having at least one thindimension such as paper or straw. If one relies on the effects ofgravity or floatation to cause movement of the solid lignocellulosicmaterial with respect to the liquid, then particle size may need to beadjusted appropriately to permit solid/liquid movement in the timeperiod of the prehydrolysis. Optimum sizes will depend on the particularlignocellulosic material used and the reactor size and construction andare readily determinable by routine experimentation.

The lignocellulosic substrate may consist of hardwood, grasses,softwood, waste paper and pulp, municipal wastes, agricultural wastessuch as straws, corn cobs, corn stover, biomass of all types, etc. andmixtures thereof. The choice of lignocellulosic substrate will depend onthe availability and cost of lignocellulosic materials.

In the present invention, the reactor generally may have a solidscontent of between about 5 and 50%, preferably about 8-30%, when thesolids are present with the liquid at the end of the prehydrolysis. Thehigher solids content are generally more desirable but the concentrationis limited by the designs of the reactor and the need for fluid to flowthroughout the solids. At the beginning of the prehydrolysis, the solidscontent may range from 0 to 100% by weight as the reactor may initiallycontain only the lignocellulosic solids or the degrading fluid.

To prehydrolyze the lignocellulosic substrate, a degrading compound isadded to the substrate either before, simultaneously or after loadingthe reactor with substrate. The degrading compound may directly degradethe lignocellulose substrate or indirectly degrade it by being convertedinto an acid or alkali or by interacting with lignocellulose so that itis more susceptible to the effects of acid or alkali.

The degrading compound to effect prehydrolysis may be either acid oralkali. Both strong and weak acids and alkali may be used provided thatthey attain the desired pH. The degrading compound may be in eithersolid, liquid or gaseous form. Generally, at least some water will bepresent during at least part of the prehydrolysis.

Representative inorganic acids include: sulfuric, sulfurous, nitric,hydrochloric, carbonic, hydrofluoric, hydrobromic, phosphoric, boric andoxy acids. Representative organic acids include: formic, acetic,pyroligneous, oxalic, malic, benzene (or other aromatic)-sulfonic,trichloroacetic, trifluoroacetic, carboxylic acids and polymers withorganic acid moieties. Representative gaseous acids include: sulfurdioxide, sulfur trioxide, carbon dioxide, chlorine, phosgene and NO_(x)where x is from 0.5 to 4. Representative gaseous alkali include:ammonia. Representative alkali include: metal hydroxides and ammonium ormetal oxides, sulfates, bisulfates, sulfites, bisulfites, carbonates andbicarbonates, P₂ O₅, urea, guanidine, amino containing compounds, saltsof weak acids and polymers containing salts of weak acids. The preferredmetals are alkali and alkaline metals to yield alkali such as sodiumhydroxide. Representative materials to form acids include: metalhalides, e.g. aluminum chloride, copper chloride, zinc chloride, tinchloride, titanium chloride, nitrophenol and chlorophenol.Representative materials to form alkali include: zinc, iron (II) oxideand hydroxy salts. Hydrates of any of these compounds which form arealso included.

Each category, as well as each compound or combination of categories orcompounds, has inherent advantages and disadvantages. Costconsiderations and the composition of the lignocellulosic material aregenerally controlling. However, certain ones have inherent advantages.For example, alkaline treatments are generally believed to be moreadvantageous at solubilizing lignin whereas acidic treatments aregenerally thought to be better at solubilizing hemicellulose. Contraryto previous belief in the prior art prehydrolysis, extreme pH is notneeded with the present invention. There is no particular advantage tousing a strong acid or alkali from a pH standpoint. Degrading compoundswhich provide nutrients or cause nutrients to form for a laterfermentation have certain advantages as these nutrients will not need tobe added later.

Gaseous degrading compounds have distinct advantages and disadvantages.Gases generally will diffuse into the lignocellulosic material faster,thereby permitting a faster reaction or the use of larger substrateparticles. Gases also may be easier to handle and require less demandingapparatus. Corrosion of the apparatus is a problem with both gas andliquid degrading agents but should be less of a problem with gasses. Onthe other hand, these gases tend to be poisonous and therefore avoidanceof leaks is a major concern.

Using gaseous degrading compounds also raises the issue of how one is toobtain liquids. To do so, one may add very hot water to the system towash away the degradation products. Alternatively, steam may be pumpedin which will condense to provide the liquid.

Alternatively steamed lignocellulosic material can produce its own acidsor alkali in situ from endogenous acidic or alkaline materials, e.g.uronic acid and acetic acid. Combinations used separately or mixtures ofacidic or alkaline chemicals may be used. One may even use combinationsfrom different categories of materials such as sulfuric acid and carbondioxide.

In this patent application, the pH altering chemicals are referred to asacids or alkali. It should be appreciated that compounds which bufferthe pH at acidic or alkaline ranges are also considered acids or alkali.

An oxidizing compound may also be added to aid in the degradation ofcomponents such as lignin. Examples of oxidizing agents include peroxycompounds (e.g. hydrogen peroxide) or peracid compounds. Otherpretreatment chemicals may also be used such as salts or metal chelatingagents.

The concentration(s) of the chemical agents to use will depend on anumber of factors such as the chemical composition or thelignocellulosic material. Additionally, the water content and theinherent buffering capacity of the substrate also regulate theconcentration and amount of degrading compound to add.

For the example of a liquid acid, such as sulfuric acid, being used asthe degrading fluid for prehydrolysis, the percolation reactor describedabove may be filled with lignocellulosic substrate and relatively mildconditions used to acid prehydrolyse the substrate. The conditionsinclude 90°-240° C., preferably 120°-180° C. at a pH of 1.0-5.5 and for10 minutes to 2 hours. The liquid drawn off is optionally furthertreated to hydrolyse any xylose oligomers. It should be noted that thepretreatment process simply removes the hemicellulose from the solids,prehydrolysis does not necessarily completely hydrolyse the oligomers.The resulting solid residue has 20-50%, preferably 30-50% of the Klasonlignin removed by the prehydrolysis. Almost all of the hemicellulose(>90% xylan removal) and relatively little (up to 25%) of the celluloseis removed by such a treatment. The resultant residue is very digestibleby cellulase.

One representative example of a gaseous acid for lignocelluloseprehydrolysis is carbonic acid. This acid can produce prehydrolyzates inthe pH range of 2-7 with 3-5 being preferred. Carbonic acid may beprepared before addition to the percolation reactor or carbon dioxidemay be injected into the reactor to prepare carbonic acid in situ.Conditions include a temperature range of 140°-240° C. with sufficientpressure for the water and carbon dioxide to remain in liquid form.

The liquid drawn off during prehydrolysis and further treated tohydrolyse xylose oligomers may then have its pH altered simply byreleasing the pressure and allowing carbon dioxide to volatilize. The pHadjusting process may be encouraged by agitation, bubbling an inert gasthrough the prehydrolyzate or adding an alkaline material. The carbondioxide release at this point and carbon dioxide produced byfermentation may be used to prepare additional carbonic acid forprehydrolysis of additional lignocellulosic substrate.

Without the flow-through reactor design, utilization of carbonic acidwould not be feasible. In closed systems, if carbonic acid were used itwould not be effective for at least one reason. Closed systems require alower pH than can be achieved with carbonic and other similar weakacids.

Another representative gaseous acid for lignocellulose prehydrolysis issulfur dioxide. After a lignocellulosic substrate is placed in thepercolation reactor, sulfur dioxide is pumped into the reactor andallowed to penetrate the lignocellulosic substrate. Steam or liquidwater is then pumped into the reactor and sulfurous acid is formed insitu. The remainder of the process and conditions are the same as forthe acidic prehydrolysis described above.

Alternatively, the reactor may already contain lignocellulosic substrateand water and then the sulfur dioxide is sparged into the reactor. Theprehydrolysis conditions of temperature and pressure may be obtainedbefore or after sulfur dioxide addition.

To further the acidic pretreatment, oxygen and/or another oxidizingagent such as permanganate may be present to convert sulfur dioxide tosulfur trioxide so that sulfuric acid will later form. A catalyst may beadded or may be part of the percolation reactor itself to convert sulfurdioxide to sulfur trioxide. Suitable catalysts include platinum orvanadium oxide. It should be noted that this reaction along with theaction of sulfur dioxide/trioxide forming dilute acid are exothermic,thereby producing some of the heat necessary for prehydrolysis.

An additional advantage of employing a solid catalyst is that itprovides physical support to the particles of lignocellulosic material.Certain lignocellulosic substrates such as newsprint tend to agglomerateor mat which prevents the even flow of gas or liquid through thelignocellulosic material. Furthermore, fluid which does flow throughsuch a mass tends to channel, thereby causing a drop in pressure and aninefficient and non-ideal distribution of fluids in the reactor.

The use of physical support materials in the reactor is not limited tosolid catalysts. Inert particles also may be used to preventagglomeration and mat formation in the reactor. Suitable inert particlesinclude, glass beads, numerous refractory materials and coarserlignocellulosic material.

Because gases are handled differently from liquids, the percolationreactor apparatus may be modified to accomplish the necessary handlingof materials. Furthermore, different degrading materials may requiredifferent control mechanisms and impart different requirements formaintaining the chemical inertness of the apparatus. These requirementsare well known and thus it would require only routine optimization toadapt the percolation reactor to any specific degrading compound(s).

A representative liquid alkaline material is sodium hydroxide. Thealkali can produce a pH of 7-14+ and prehydrolysis may occur fromambient temperature (about 10°-30° C.) to 250° C. The remainder of theprocess is the same as for acid prehydrolysis except for altering the pHtoward neutral will require an acidic material.

A representative gaseous alkaline gas is ammonia. This material also hasthe advantage of constituting an essential nutrient in a laterfermentation. The conditions are the same as above and the equipment mayneed modifying for gaseous acid compound(s) as noted above.

The liquid flow volumes through the reactor are preferably low in orderto keep the concentration of removed sugars high. Generally, the entireflow volume will range from about 1 to 5 total void volumes, preferablyabout 2 void volumes. While this may produce about twice the liquid of aclosed prehydrolysis system, the sugar concentration may be roughly thesame after hydrolysis of oligomers because significantly more sugar isremoved by the present invention. For example a typical batchprehydrolysis system may produce a liquor having about 4-5% sugarconcentration which after washing produces about 3% sugar concentration.By comparison, the present invention may also produce about 1-4% sugarconcentration in the liquor.

The pressure during prehydrolysis may be simply the existing pressurewhen heating aqueous solutions to the desired temperature or slightlymore than the pressure generated from water vapor at the particulartemperature (steam pressure). The prehydrolysis reaction occursprimarily in a liquid phase and at least at the end of the prehydrolysisreaction, the degrading fluid is to be a liquid. High overpressures areneither necessary nor particularly desired.

The duration and temperature of the prehydrolysis step will varydepending on the lignocellulose substrate. Furthermore, the compositionof the prehydrolyzate fluid eluting from the reactor will vary dependingon the substrate used. Generally conditions are optimized so thatcellulose is not substantially degraded by prehydrolysis. This does notmean that no glucose will be removed from the lignocellulose. Hardwoodsundergoing prehydrolysis will typically loose about 5 to 25% glucoseduring prehydrolysis. Softwoods typically will loose more, in the rangeof about 10 to 30% glucose during prehydrolysis.

Prehydrolysis may end with a pulse of water to wash the residue andremove degrading compounds from the solids. The wash water may berecycled to the prehydrolysis reactor by adding make-up dilute acid oralkali or it may be added to the prehydrolyzate liquor. When thistechnique is not used or is insufficient to adjust the pH to anacceptable range for cellulase, a small amount of neutralizing solutionor buffer may be added to adjust the pH. This step may be performed inthe prehydrolysis reactor or after the solids have been removed. Theresidue is particularly well adapted to enzymatic digestion by cellulaseat this stage.

The liquor from the prehydrolysis reactor contains some soluble pentoseand hexose, lignin and oligomers of pentoses. The oligomers are thenhydrolysed to monomers, or at least to dimers or trimers, by anyconventional technique. The simplest may be to simply hold theprehydrolyzate liquor at the prehydrolysis temperature in a holdingvessel. Since this temperature may be too high, the liquor may beflashed by releasing the pressure in the holding vessel. Conditions inthe holding vessel are such that oligomers are hydrolyzed whiledegradation is minimized. Hemicellulases are also known and may be usedalone or in combination with the holding treatment if so desired.

The conditions during the temperature hold may be used for otherpurposes in addition to hydrolyzing xylose, or other sugar, oligomers tomonomers. For example, the conditions may be controlled to enhancerecondensation or precipitation of lignin. Precipitated lignin is easilyremoved and may be used to produce other chemicals, used in or on soilas a mulch etc. or burned as fuel.

Oligomers of xylose are generally not readily fermentable by mostmicroorganisms. For example, ethanologenic strains of Klebsiella oxytocacan utilize xylose dimers and trimers but not oligomers of xylose withhigher degrees of polymerization. See Burchhardt et al, Applied andEnvironmental Microbiology 58: 1128-1133 (1992).

After sufficient hemicellulose has been hydrolysed in the hydrolyzateliquor, the pH of the fluid is adjusted to a pH acceptable forfermentation. If the degrading chemical is sulfuric acid, the preferredpH adjusting compound is calcium hydroxide or calcium oxide. Thismaterial not only neutralizes the sulfuric acid but it produces gypsum,its half hydrate or other forms, which is insoluble in water. Theprecipitated gypsum is then separated to remove it from the process. Thetemperature is at least about 90° C. and preferably is at least about100° C. during the separation unless the fluid is under pressure. Highertemperatures under pressure such as 120° C. and above or theprehydrolysis or temperature hold temperatures may also be used. Thus,this neutralization and gypsum removal technique is broadly applicableto any chemical process.

Gypsum, unlike most compounds, is less soluble in the aqueous solutionat higher temperatures than at room temperature. Separation may bepreformed with a centrifuge, a filter or simply allowing the solidgypsum to settle in a settling vessel. Therefore, pH adjustment and/orat least separation of gypsum is preferably performed when the fluid ishot. This technique may also be applied to the neutralization of othersulfuric acid solutions such as that produced by acid hydrolysis ofcellulose. The gypsum may be recovered as a useful chemical such as inthe production of plaster of paris.

Previously, gypsum formation has resulted in fouling and numerousdifficulties downstream. For that reason, previous prehydrolysistechniques have used cocurrent reactors as a way to reduce the problemswith gypsum coating and clogging the apparatus. The present inventionmore effectively removes gypsum and therefore avoids previous problemsand permits one to use a different apparatus.

If the degrading chemical is carbonic acid, the pH adjustment occurs ina different manner. Instead of adding a chemical, one can simply reducethe pressure in the system and carbonic acid will be removed byvolatilizing carbon dioxide. The carbon dioxide may be recovered fromthis stage and from the fermentation and reused earlier in the processto prepare carbonic acid. This technique has certain advantages inavoiding or reducing the cost of degrading and neutralizing chemicals.To further aid in carbon dioxide removal, an alkali may be added to theprehydrolyzate liquor and/or an inert gas may be pumped through thefluid to encourage removal of carbon dioxide gas.

After the pH has been adjusted to an acceptable range, the concentrationof sugars in the prehydrolyzate liquor may be measured. If theconcentration is too high, the liquor is diluted. If the concentrationis too low, the solution is concentrated by any standard dewateringtechnique which will not adversely affect the sugars. The sugars may berecovered and purified or utilized without further purification beingnecessary.

The liquor is then ready to be fermented to an organic compound such asan alcohol (ethanol, etc), a ketone (acetone, etc.), a carboxylic acid(butyric acid, etc.) etc. The liquor may be supplemented with othernutrients, its physical and chemical properties (pH, temperature,concentrations, etc.) measured and modified if necessary and fermentedby a microorganism capable of utilizing the sugars in the liquor toproduce a desired organic compound. This technique is known per se andis thoroughly described in the fermentation art.

After the fermentation has produced the desired organic compound, thecompound is removed from the fermenter by techniques known per se suchas distillation. The fermentation and removal each may be continuous,batch or fed-batch.

The solid residue remaining after prehydrolysis may be removed and haveits pH adjusted in a similar manner to a pH suitable for cellulasedigestion of cellulose. Alternatively, one may wash the solids withwater, to reduce or eliminate the need for neutralization. This washingstep may be performed at the end of prehydrolysis while maintaining thetemperature and pressure, or it may be performed after prehydrolysis hasbeen completed. The wash water may be reused, discarded or added to theprehydrolyzate liquor as desired. The pH and solids content may bemeasured and adjusted to optimize for cellulose digestion by cellulase.

By using this percolation pretreatment system and "washing out"hydrolyzed acid-labile lignin, a substrate is produced which has beensignificantly delignified and is very amenable to enzymaticsaccharification using a cellulase enzyme system. A "significantlyreduced amount" of lignin constitutes the amount needed to enhancedigestibility of the lignocellulosic substrate by the enzyme cellulase.The "enhanced digestibility" is enhanced over the previous techniquesresulting in 5 to 15% removal of lignin. For the purposes of this patentapplication, a "significantly reduced amount of lignin" is greater thanabout 20% removed.

Cellulase is then added to the pH adjusted residue and the cellulosedigested to sugars. This process is carried out in a manner known perse. Any of the known cellulases or cellulase complexes may be used.Typically the digestion will be carried out for one to three days at atemperature optimal for the cellulase. The sugar containing solution isseparated from the residues by, for example, filtration, sedimentationor centrifugation. The sugar solution may be recovered as sugars or itmay be fermented in a manner known per se to produce a desired organicchemical.

The fermenting microorganism may be the same as was used to ferment thehydrolyzate liquor. However, because cellulose digestion primarilyproduces glucose, a much wider variety of microorganisms may be used toproduce an even wider assortment of organic compounds. The residuedigest may be fermented in any manner known per se to utilize glucose.If so desired, the residue digest may be mixed with the hydrolyzateliquor before or during fermentation.

As an alternative to separate cellulase digestion and fermentation, bothreactions may occur simultaneously in "simultaneous saccharification andfermentation", a technique known per se. Any fermentation which isoperable in conditions suitable for cellulase is acceptable. Theconditions and concentrations in simultaneous saccharification andfermentation (pH, temperature, etc.) may be measured and adjusted to beoptimized for either saccharification or fermentation or for overalloptimization. The conditions may be changed as the process progresses.The prehydrolyzate liquor may be added to the simultaneoussaccharification and fermentation if desired.

One may combine the fermentation of the prehydrolyzate liquor and thefermentation of the sugars from the solids simultaneously. Thiscombination has certain advantages and disadvantages. Whether to use acombined fermentation would depend on the composition of the feedstockand the economics of the particular plant and local area. Advantagesinclude the use of fewer fermentation vats. Also, cellulase would becarried over into the fermentation and many cellulase preparationscontain xylanase activity. This would serve to degrade any residualoligomers of xylose. Disadvantages include the need for specializedmicroorganisms for fermenting pentoses whereas glucose can be fermentedby many microorganisms such as yeast.

Because the prehydrolysis used in the present invention removes agreater amount or percentage of lignin and hemicellulose from thelignocellulosic material, the residue is more easily digested withcellulase. This improved result is reflected by four desired properties.

With less lignin and hemicellulose binding to the cellulose fibers,greater contact between cellulase and cellulose is achieved. This isreflected in a greater speed of digestion. Secondly, because lesscellulose is hidden behind a layer of lignin and hemicellulose, a higheroverall digestibility of the substrate and higher yield of sugar isobserved. Thirdly, cellulase binds to lignin. With less lignin present,less cellulase binds to lignin and therefore the cellulase added appearsto have a greater activity in use. Accordingly less cellulase is neededto perform the digestion. Fourthly, the significantly reduced lignincontent remaining in the solid residue translates into reduced lignin inthe fermenter. This permits one to use a smaller fermenter, less powerto mix it and results in less lignin clogging up all of the down streamsystems. All of these advantages result in significant cost reductionsin the overall process of producing sugars or fermented organiccompounds from lignocellulose.

Unlike previous prehydrolysis techniques, the present invention permitsone to use more than one set of conditions during a singleprehydrolysis. If fluid is passed through the solids continuously or atleast at the end of each set of conditions, then the prehydrolysis maybe optimized to solubilize different materials at different times. Forexample, with the previous prehydrolysis systems, one would need to endthe prehydrolysis, remove the material from the reactor, wash the liquidfrom the solids, return the solids to the same or a differentprehydrolysis reactor and perform a separate prehydrolysis underdifferent conditions to obtain a second liquid fraction. With aflow-through reactor of the present design, one can change thetemperature, pressure, pH and chemical additions during the sameprehydrolysis step while removing different fractions of solubilizedliquid.

Different fractions of liquid produced by a varying prehydrolysis may bepost-treated differently and optimized for their separate chemicalcomposition. Closed prehydrolysis systems do not permit such a variationin conditions with removal of the fluid between different conditions. PHwould be almost impossible to control or vary in a closed system.

The present invention includes the changing of at least one condition atleast once during a single prehydrolysis step. The prehydrolyzate liquoris withdrawn during or at the end of each set of conditions. Thecondition(s) may be changed any number of times and may even becontinuously changed.

Using plural conditions during prehydrolysis has at least three distinctadvantages over conventional prehydrolysis. First, differentlignocellulosic substrates may be treated differently as appropriate tooptimize results. This is particularly advantageous when one considers acontinuous addition of lignocellulosic substrate to the prehydrolysisreactor. The substrate may be analyzed for its physical properties andchemical composition as it arrives and optimal prehydrolysis conditionsadjusted accordingly.

Secondly, fluid travel in a flow-through reactor cannot be ideal eventhough this is desirable. While experiments exemplified below show nearideal distribution of fluid throughout the reactor, in practice it maybe very difficult to maintain an ideal distribution. Accordingly, it isdesirable to change the conditions in the prehydrolysis reactor tocompensate for non-ideal conditions. The present invention permits sucha change by sensing conditions inside the prehydrolysis reactor andliquid or solid products leaving the reactor and from that dataadjusting the conditions in the prehydrolysis reactor to compensate fornon-ideal distribution.

Thirdly, one can periodically or continuously monitor the products orconditions inside or emitting from the prehydrolysis reactor as ameasure of the effectiveness of the prehydrolysis reaction. Should themeasurements vary from what is considered optimal, the conditions withinthe prehydrolysis reactor may be changed accordingly. This type ofsystem lends itself to constant monitoring and control by a feedbackloop with a computer.

The harshness or severity of the prehydrolysis treatment is a functionof the temperature, time, pressure and concentration and type ofchemical, particularly the pH generated by the chemical. The combinationof these features are critical to the degree of prehydrolysis and adecreased value for one can frequently be compensated by an increase inone of the other parameters. The result is the kinetic concept of acombined severity parameter. This idea is certainly not new to chemicalengineering as time and temperature have long been recognized asadjustable in opposite directions to usually yield the same result.

In the field of lignocellulosic prehydrolysis and fractionation of woodcomponents, elaborate equations for calculating a "combined severityparameter" have been used. See Chum et al, Applied Biochemistry andBiotechnology 24/25: 1-14 (1990). The combined severity parametercalculations have even been used to justify radically differentparameters such as using simply water, presumably close to pH 7, with avery high temperature and pressure. See Mok et al, Ind. Eng. Chem. Res.31(4): 1157-1161 (1992).

A significant benefit from the present invention is the use of lesssevere conditions with a lower combined severity parameter resulting ingreater component separation using the method of the invention ascompared to previous prehydrolysis techniques. Furthermore, when using alower combined severity parameter, greater hemicellulose removal andreduced production of unwanted breakdown products (furfural, tars, etc.)was observed as compared to more severe conditions in a simple reactorwith separation of liquid after prehydrolysis was completed and reactorcooled.

Another significant advantage to the present invention is the use of apH which is less extreme. Previous attempts at acid prehydrolysis at160° C. in a close reactor have required a very low pH. Some have evenspeculated that a pH above 1.5 is inoperable because of the very longtime needed to achieve substantial prehydrolysis at this temperature.With the present invention, a pH in ranges as high as 3-4 at atemperature of 160° C. can produce a digestible solid substrate.Therefore, using a percolation reactor in the process of the presentinvention has permitted substantially less acid to be required, agreater choice of acids, and less demanding equipment to handle thereaction.

In the present invention, one is even able to use prehydrolysisconditions which are not acceptable for a closed batch prehydrolysisprocess. Because of the use of less severe conditions, less unwanteddegradation products such as furfural and tars are formed during theprehydrolysis. This is particularly advantageous when the results are tobe fermented as furfural compounds and tars are toxic to manymicroorganisms and in sufficient concentrations inhibit fermentation.

The use of less severe conditions provides a number of additionaladvantages such as using less demanding equipment, numerous costadvantages and most importantly, permits the use of a less extreme pHand permits the use of different degrading compounds. Many of thesedegrading compounds would not be effective in closed prehydrolysissystems because they do not generate extremely severe conditions.

One example of a particular apparatus which may be used in the processof the present invention is a screw device which conveys solids by dragforces while liquids move in the opposite direction by the forces ofgravity. The design may be a conventional intermeshing twin screwdevice, an Archimedes screw or another screw device which use the actionof drag to move the solids and gravity to move the liquids may be used.

This screw device is maintained under prehydrolysis conditions. It maybe the primary prehydrolysis vessel or it may simply receive materialfrom a prehydrolysis vessel through its inlet port which was already atleast partially prehydrolyzed. The screw device effects counter currentsolid/liquid separation while the material is under prehydrolysisconditions. Therefore, the screw device performs the same function of aflow-through prehydrolysis reactor without actually pumping anyadditional fluid into the reactor.

The screw device may be vertical or inclined. The solid-liquid slurry ormixture is added through an inlet port in a barrel of the screwsomewhere between the ends. The solid materials are moved to the topwhile liquids drain to the bottom. Appropriate discharge ports forsolids and liquids are provided.

The general construction of similar screw devices is well known andcommercially available. Examples include Holstead et al, U.S. Pat. No.3,859,217 and Duchateau et al U.S. Pat. No. 4,311,673. Screw deviceshave even been used in enzymatic digestion of materials for ethanolproduction, Hayes, U.S. Pat. No. 4,326,036 and soaking cellulosicmaterial before digestion in a gas phase, Richter, U.S. Pat. No.3,532,594. However such screw devices are generally not adapted or ableto maintain the conditions appropriate for lignocellulose prehydrolysiswhich are mentioned above. Furthermore, screw devices have notheretofore been used for solid/liquid phase separation of reactionproducts in any chemical reactor.

Slots, grooves, dimples and the like may be present on the barrel orscrew blades to assist in the conveyance of the solids. The screw bladesmay also contain pores or screen-like portions which allow liquids topass through the screw while retaining the solids. To further separateliquids from the wet solids, the top portion of the screw may havethreads which become progressively closer together so that the solidsmay be squeezed to force out more liquid as the screw rotates. Washwater may be added to the screw through a port in the barrel at anylocation, preferably at a level above the inlet port.

Solids finally produced at the top of the screw are transported away bya scraper, auger or the like. Liquids produced at the bottom of thescrew are removed. If the screw is maintained under pressure, highpressure pumps may be used to remove the solids and liquids.Alternatively, the solids and liquids may be stored in separate holdingvessels until further use. As with the general flow-through reactorsdescribed above, it is important for the screw to effect solid/liquidseparation while the mixture is still hot.

The invention can be better illustrated by the use of the followingnon-limiting examples, all of which related to the acid prehydrolysis oflignocellulosic material.

EXAMPLE 1

A lignocellulose containing substrate used in these Examples is Populuseugeneii (hybrid poplar) DN 34 which was provided by the University ofMinnesota at Crookston. It was harvested in the fall of 1992, manuallydebarked, and coarsely chipped using a Formost mobile knife chipper("Brush Bandit"). The chips were milled further using a laboratory knifemill (Thomas-Wiley laboratory mill, Arthur H. Thomas Co., Philadelphia,Pa.) equipped with a 1-mm rejection screen. Milled material was furtherseparated into a -60 to +80 mesh (0.18-0.25 mm) fraction by using aportable sieve shaker (Tyler Industrial Products, Mentor, Ohio) equippedwith USA Standard Testing Sieves. This material constituted the"biomass" for the experiments performed in the examples.

A liquid cellulase preparation used in these Examples (Genencor Laminexcellulase, San Francisco, Calif.), was stabilized by the addition ofglycerol and stored at 4° C. until it was used. The specific activity ofthe enzyme was approximately 64 international filter paper units(IFPU)/mL (22). β-glucosidase activity in this preparation wasapproximately 82 international units (IU)/mL (22). Fungal β-glucosidase(250 IU/mL 22!, Novozyme 188, NOVO Lab Inc., Wilton, Conn.) was used tosupplement the cellulase preparation such that the ratio ofβ-glucosidase to cellulase was 3:1 in all Examples. The yeast used inthe simultaneous saccharification and fermentation (SSF) Examples wasSaccharomyces cerevisiae D₅ A. This strain is described in Spindler etal, Biotechnology Letters, 14: 403-407 (1992). The remaining chemicalswere purchased from national laboratory supply houses. Cellulose powder(α-cellulose), used as a control substrate, was obtained from SigmaChemical Co. (St. Louis, Mo.).

The design of the percolation reactors used in these Examples resemblespressure chromatography columns with valving at both ends and dispersionfrit at the entrance followed by a retention frit. A retention frit wasalso installed at the exit.

The apparatus used included a percolation reactor that was of sufficientdimensions so as to hold enough raw and pretreated biomass foranalytical work. A reactor 2 in. (51 mm) in length by 1 in. (25 mm) indiameter was used. The particle size, -60 mesh to +80 mesh (0.18 to 0.25mm), was chosen to minimize dispersion of the acid catalyst in theflow-through reactor. Before dilute-acid pretreatment experiments couldbegin, a flow characteristic study was initiated to quantify the flowbehavior of the catalyst as a function of temperature and flow rate.Once the flow characteristics were quantified, the pretreatmentexperiments were conducted. The quality of the pretreated residue wasassessed by both enzymatic saccharification and the conversion of theglucan to ethanol by the SSF protocol.

A threaded hole is located at the midpoint of the reactor length throughwhich a thermocouple is installed to monitor temperature. Thethermocouple head may be bent to be located at almost any point in theinterior of the reactor. For all experimental runs in these examples,the thermocouple head was located at the center of the reactor.Carpenter 20Cb-3 stainless steel was used as the material ofconstruction for the reactor. The dispersion frit (1-in. 25 mm!diameter, 2 micron pore size, made of titanium) and the retention frits(1-in. 25-mm! diameter, 60 micron pore size, made of Inconel) wereobtained from Mott Metallurgical, Farmington, Conn. Titanium tubing(1/16-in. 1.6-mm! outside diameter×0.03-in. 0.8-mm! inside diameter)used to connect the reactor with other components of the system wasobtained from Anspec, Ann Arbor, Mich. The 2-in. 51-mm! bar stock (usedfor the head plates of the reactor) and the 1-in. 25-mm! ID tubing (usedfor the body of the reactor), which were both made of Carpenter 20Cb-3stainless steel, were obtained from Carpenter Technology, Dallas, Tex.,and Marmon Keystone, Denver, Colo., respectively. The three-wayHastelloy C valves were obtained from Valco Valve Corporation, Houston,Tex. The third port of the three-way valves was not used for theseexperiments. The reactor was fabricated and assembled by FalconFabrication, Arvada, Colo.

A schematic of the flow system and equipment design is seen in FIG. 2.For all flow characteristic studies and dilute-acid pretreatment runs,the following protocol was used. The reactor was charged with 3.80 g(3.53 g bone dry basis) of ambient-air-dried biomass and the reactorhead plates bolted shut. Once the reactor was closed, the inlet line wasattached to the water line from a high performance liquid chromatograph(HPLC) pump (Beckman Instruments, model 110B, Fullerton, Calif.) and theexit line was left open to the atmosphere. Water was pumped through thereactor at ambient temperature at 1.1 mL/min for at least 4 h to ensuretotal wetting of the biomass. The reactor was then submerged in a 90° C.sand bath (SB series General Laboratory Fluidized Bath equipped with atemperature controller, Cat #W3280-3, VWR Scientific, Denver, Colo.)with water being pumped through the reactor at 1.1 mL/min until thecenter of the reactor reached 88° C. The reactor was then heated for anadditional 10 min to allow the trapped air in the wood pores to escape.The reactor temperature was then changed either for flow characteristicsexperiments or pretreatment experiments as described below.

EXAMPLE 2

The total void volume of a water-saturated, deaerated, packed reactorwas determined by quantitatively removing the entire contents of thereactor and immediately recording the weight, followed by drying thereactor contents at 105° C. The weight difference between the wet anddried reactor contents is a measure of the water present in the reactoror of the total void volume of the reactor, which was determined to be21.4 mL.

In order to define the prehydrolysis time in the reactor, the flowcharacteristics in the reactor were examined to determine how theobserved flow varies from ideal plug flow. The method of residence-timedistribution (RTD) determination of Smith et al, Chemical EngineeringKinetics, 3rd. ed. McGraw-Hill, N.Y. (1981), in a homogeneous reactoroperated isothermally was employed. In this part of the investigation,conditions (of tracer and temperature) were selected so that reactionsdid not occur. The RTD method assumes that the linear axial velocity, u,and the tracer concentration are uniform across the diameter. In thepacked-bed percolation reactor, It was further assumed that the totalvoid volume is distributed evenly throughout the length of the reactor.Accordingly, the deviation from ideal plug-flow conditions can bedetermined from the tracer concentration in the effluent in response toa step change in the feed tracer concentration. The governing equationis ##EQU1## where C and C_(o) are the tracer concentrations in theeffluent and feed, respectively; L is the reactor length; D_(L) is theaxial effective diffusivity; θ is the elapse time after the step changein the feed tracer concentration; Θ is the mean residence time; and erfis the error function. See Abramowitz et al, Handbook of MathematicalFunctions, 10th printing, National Bureau of Standards (1972). As such,the response in the effluent, C/C_(o), is a function of D_(L) /uL (thereciprocal of the Peclet number), and the greater the group D_(L) /uL,the greater the flow in the reactor deviates from ideal plug flow.

For the RTD studies, the reactor was charged with biomass and deaeratedas described above. The reactor inlet was connected to the HPLC pump andthe exit port was connected to a RI detector (Altex model 156). Threereservoirs were available for liquid flow; 10 mM sodium chloride,deionized water, and 0.4 wt % sulfuric acid. At time zero, either theNaCl or H₂ SO₄ reservoir was brought on line and replaced the waterreservoir for the Step-change tracer experiments. A strip chart recorder(Recordall, Fisher Scientific, Denver, Colo.) was connected to the RIdetector, which responded to both the NaCl and H₂ SO₄ solutions. For the140° C. tracer studies, the reactor was submerged in a 140° C. sand bathand connected on line through the RI detector with a valve to thereceiving column (Omni glass chromatography column assemblies rated at150 psig 1,030 kPa gauge!) closed. Once the reactor reached 140° C.,nitrogen gas was used to charge the receiving column and match thereactor pressure. When the entire system was at equal pressure, thevalve was opened. The entire transient response in the reactor effluentafter the feed was step-changed to either NaCl or H₂ SO₄ was recorded.The experimental reproducability was verified by conducting two runs ontwo different days using two different biomass packings.

Because of the height to diameter ratio of the reactor channeling couldhave been a problem if the dispersion frit was ineffective. A controlresidence time distribution (RTD) experiment was run in which anentrance head plate was drilled out forming a conical shape to allow theentering liquor to disperse over the entire surface area of the biomass.If channeling was a problem, the RTD function for the non-conical headplate would have shown more non-ideal flow characteristics than thedrilled out head plate. Experiments revealed this to not be the case.Therefore, channeling was not a problem.

EXAMPLE 3

In this example, a two-temperature pretreatment using a percolationreactor packed with the hybrid poplar wood flour biomass was used. Inorder to better define the experimental conditions that would give highxylose equivalent yields, a mathematical modeling of the process usingthe two-temperature pretreatment of hybrid poplar xylan was used. It hasbeen demonstrated through mathematical simulations (See Kim et al,Applied Biochemistry and Biotechnology, (1993) accepted for publicationfor mathematical modeling) that after the biomass is cooked attemperatures between 135°-150° C. using 0.73 wt % sulfuric acid forresidence times necessary to hydrolyse 60% of the xylan, a step-changeincrease in prehydrolysis temperature of between 25° and 35° C. tohydrolyse the remaining xylan results in maximum yields of xylose in theprehydrolyzate. Furthermore, using 170° C. as the upper prehydrolysislimit, maximum yields of xylose are obtained by using 140° C. as thelower prehydrolysis temperature and residence times of 34 minutes, 20seconds and 21 minutes, 51 seconds at the lower and higher temperatures,respectively. A total volume of hydrolysis liquor equaling two totalvoids was calculated to give a maximum xylose equivalent yield. Althoughthe wood substrate used in the mathematical modeling had a slightlydifferent chemical composition than the biomass used in the presentexamples, the above residence times and temperatures were used in thepresent protocol and considered to be appropriate.

The reactor was charged with biomass and deaerated as described above.Immediately following deaeration, the reactor was connected to acollection system with the valve closed. The reactor was then submergedin the 140° C. sand bath. Once the reactor reached the prehydrolysistemperature, the system was pressure-equalized as described above, 0.73wt % sulfuric acid was pumped to the reactor at the rate of 8.8 mL/min(linear flow rate of 2.09 cm/min) for 1.5 min (the time for the acid tofirst appear at the exit end of the reactor), and the effluent liquordiscarded (all subsequent prehydrolyzate was collected for analysis).The acid was then pumped at 8.8 mL/min for an additional 2.5 min tototally saturate the reactor with acid (which was determined from flowcharacteristics studies). The pumping rate was then adjusted to equaljust under one total reactor void volume over the desired prehydrolysistime to hydrolyse approximately 60% of the xylan (flow rate=0.62 mL/minfor 30 minutes, 15 seconds). The reaction was quenched by pumping waterto the reactor at the rate of 8.8 mL/min for a total of 6 minutes, 10seconds which was the time to completely wash all the acid out of thereactor. The pump was then shut off and the valve closed. With thereactor remaining in the sand bath, the temperature was raised to 170°C., the system pressure was equalized, the valve was opened, and theliquor pumping sequence mentioned above was repeated (except that thepumping rate and time used to send just over one total void volumethrough was 1.35 mL/min for a total of 17 minutes, 45 seconds). Afterquenching the reaction as described above, the reactor was cooled toambient temperature and the solid contents collected in a glass-sinteredfunnel of medium porosity. The solid residue was then weighed andchemically analyzed; the combined prehydrolyzate (from 140° and 170° C.prehydrolyzates) was analyzed for xylose and other components.

EXAMPLE 4

The dry weight of all solids and the ash content of the native feedstockwere determined by standard methods (Official Test Methods, TAPPI,Atlanta, Ga.). Lignin and other acid-insoluble components weredetermined as Klason lignin by standard methods. Moore et al, Proceduresfor the Chemical Analysis of Wood and Wood Products, (1967) USDA ForestProducts Laboratory, Madison, Wis. Acid-soluble lignin was determined byusing an aliquot from the Klason lignin filtrate by standard methods.Technical Association of the Pulp and Paper Industry Standard Method T250, TAPPI, entitled "Acid-soluble Lignin in Wood and Pulp". Uronicacids, acetyl groups, and furfural were determined as described inTorget et al, Applied Biochemistry and Biotechnology 24: 115-126 (1990).The carbohydrate composition of biomass solids was determined by amodification of the two-stage sulfuric acid hydrolysis (Moore et al,Procedures for the Chemical Analysis of Wood and Wood Products, (1967)USDA Forest Products Laboratory, Madison, Wis.) followed bydetermination of monomeric sugars by ion-moderated partition (IMP)chromatography (the slight modification was the use of a 2-h incubationof the substrate in 72 wt % sulfuric acid instead of a 1-h in order tosolubilize the glucan completely). The prehydrolyzates (prehydrolyzateis defined as the liquid phase resulting from an acid pretreatment run)obtained from pretreatment experiments had their pH raised by addingcalcium carbonate and filtered. The carbohydrates could then be analyzeddirectly. If the presence of oligomeric sugars was suspected in theneutralized filtrate, the prehydrolyzates were adjusted to 4 wt %sulfuric acid and autoclaved at 121° C. for 1 h (See Moore et al,supra), neutralized, and analyzed by IMP chromatography using Aminex HPX87XP and HPX-87C columns (Bio-Rad, Richmond, Calif.), deionized water aseluant, and refractive index (RI) detection.

The results of the hydrolysis showed that 13.9% of the glucan wassolubilized. Furthermore, 92.0±1.6% of the xylose was recovered and90-100% of the galactose, manose and arabinose were recovered. Theamount of degradation products produced was small. Furfural was 2% ofthe xylan, hydroxymethyl furfural was 0.4% of the glucan and 0.04% ofthe prehydrolyzate was acetic acid.

EXAMPLE 5

The amount of xylose as compared to oligomeric xylose was determined inthe prehydrolyzate. A sample of the prehydrolyzate was passed through aHPLC column and the peaks were measured. The results are showngraphically as FIG. 3a. The peak at 13.74 minutes represents xylosewhich was given a relative height of 180.

The hydrolyzate was hydrolysed with 4% sulfuric acid to hydrolyse alloligomeric carbohydrate to their monomers. A sample of this was passedthrough the same HPLC column and the peaks were measured. The resultsare shown graphically as FIG. 3b. The peak at 13.70 minutes representsxylose which was given a relative height of 320.

This data demonstrates that approximately 44% of the hydrolysed xylan inthe prehydrolyzate was present as oligomers.

EXAMPLE 6

Enzymatic hydrolysis was performed in batch mode at 50° C., pH=4.8 usinga 0.05M sodium citrate buffer, in gently rotated 20-mL glassscintillation vials at approximately a 45° angle as previouslydescribed. Grohmann et al, Biotechnology and Bioengineering Symposium,15: 59-80 (1985), Grohmann et al, Biotechnology and BioengineeringSymposium, 17: 135-151 (1986) and Torget et al, Applied Biochemistry andBiotechnology, 24: 115-126 (1990). Cellulase enzyme loading wasapproximately 42 IFPU/g cellulose and supplemented with fungalβ-glucosidase at approximately 126 IU/g cellulose. This level ofcellulase loading has been shown previously to be at saturating levelsof activity when using α-cellulose as a standard. See Grohmann et al,Biotechnology and Bioengineering Symposium, 15: 59-80 (1985). This isabove the IFPU/g cellulose used in the SSF Examples described here andbelow.

In addition to testing the pretreated substrate for the efficacy of thepretreatment in terms of rates and the extent of enzymaticsaccharification, an SSF protocol was used to give additionalinformation on the quality of the pretreated substrate as to its rateand the extent of convertability of its glucan content to ethanol.Extensive research has demonstrated that SSF, the simultaneoussaccharification (hydrolysis) of cellulose to glucose and fermentationof glucose to ethanol, improves the kinetics of biomass conversionthrough circumvention of enzyme inhibition by hydrolysis products,minimization of contamination risk because of the presence of ethanol,and reduction of capital equipment requirements. The kinetics of theprocess are described by Spindler et al, Applied Biochemistry andBiotechnology 28/29: 773-786 (1991).

Shaker flask SSFs were carried out in 250-mL flasks outfitted withstoppers constructed to vent CO₂ through a water trap as previouslydescribed by Spindler et al, Applied Biochemistry and Biotechnology28/29: 773-786 (1991), with minor modifications. The cellulasepreparation was employed at a concentration of 25 IFPU/g cellulose forboth the standard α-cellulose and the pretreated poplar wood andsupplemented with β-glucosidase at approximately 3 IU β-glucosidase to 1IFPU cellulase. This cellulase loading translates into 21.4 IFPU/gnative bone-dry poplar hybrid. Ethanol concentrations in thesupernatants were measured by gas chromatography as previously describedby Spindler et al, supra.

The percentage of cellulose digested by cellulase for the prehydrolyzedproduct of the present invention is represented by circles in FIG. 4a.By comparison, α-cellulose was digested under identical conditions as acontrol and the data is represented by triangles in FIG. 4a. FIG. 4bdisplays the percentage of ethanol which is produced as compared to thetheoretical maximum from fermentation of the given amount of cellulosewhen subjected to a simultaneous saccharification fermentation. Again,the prehydrolyzed product of the present invention is shown as circlesand α-cellulose control is shown as triangles.

From the data, one can see that the cellulosic product produced in thepresent invention is more readily digestible and fermentable than evenplain cellulose alone as represented by α-cellulose. Therefore, itappears that the prehydrolyzed solids have greater digestibility whichmay be due to characteristics other than simple removal of hemicelluloseand lignin.

EXAMPLE 7

To a percolation reactor, which was made of Carpenter 20Cb-3 stainlesssteels and 2 in. (51 mm) in length by 1 in. (25 mm) in diameter, wasadded 3.80 g(3.53 g bone dry basis) of ambient-air-dried poplar hybridDN 34 wood meal (-60 mesh to +80 mesh 0.18 to 0.25 mm!). Once thereactor was closed, the inlet line was attached to a water line from ahigh performance liquid chromatograph (HPLC) pump with the exit linebeing left open to the atmosphere. Water was pumped through the reactorat ambient temperature for at least 4 h to ensure total wetting of thebiomass. The reactor was then submerged in a 90° C. sand bath with waterbeing pumped through the reactor until the center of the reactor reached88° C. The reactor was then heated for an additional 10 min to allow thetrapped air in the wood pores to escape. The reactor temperature wasthen changed to 140° C. and a volume equal to one total void (21.4 ml)of dilute sulfuric acid solution (0.73 wt %) was pumped through thereactor and collected for 30 minutes solubilizing the"easy-to-hydrolyze" carbohydrate and concomitantly some Klason lignin.The reactor temperature was then changed to 170° C. and a volume of theacid solution equal to one total void (21.4 ml) was then pumped throughthe reactor and collected for 20 minutes to solubilize the"hard-to-hydrolyze" carbohydrate fraction and again concomitantlysolubilize some Klason lignin. The reactor contents were then flushedwith a volume of water equal to three total voids (64.2 ml) at 170° C.The collected liquor fractions which were combined, and the solidfraction were then chemically analyzed.

The chemical analysis of the pretreated solid substrate indicated that44% of the Klason lignin in the starting biomass had been solubilized,while 94% of the starting xylan and 16% of the starting cellulose weresolubilized. Similar data resulted when only one prehydrolysis conditionof 170° C. for 20 minutes was used when comparing removal ofhemicellulose and lignin from a lignocellulosic material.

EXAMPLE 8

To demonstrate that a flow-through reactor is more effective atseparation of hemicellulose and lignin from cellulose, applicants ran apair of experiments in exactly the same percolation apparatus. Thereactor was filled with the same amount of lignocellulosic feed, dilute(0.73%) sulfuric acid was added, and the reactor was heated to 170° C.for 10 minutes. Fluid was not pumped through the reactor.

In one experiment, 170° water was pumped through the reactor at the endof the heat treatment. This was called the "hot flushed" experiment. Inthe other set of experiments, the reactor was cooled to ambienttemperatures and its contents emptied, followed by washing the reactorcontents by water at ambient temperature. This was called the "coldflushed" experiment. The solid residue was weighed wet, dried andweighed dry and the amount of each component was determined as above.

    ______________________________________               Feed  Cold Flushed                                 Hot Flushed    ______________________________________    "Wet" Residue (g)                 5.5     9.9         10.7    % Total Solids                 92.86   30.98       26.73    Dry Residue (g)                 5.11    3.07        2.86    ______________________________________

    ______________________________________    Dry Weight Percent            Feed    Cold Flushed                               Hot Flushed    ______________________________________    Glucose   48.56     65.12      68.54    Xylose    18.24     2.04       1.46    Arabinose 2.01      0.78       0.67    Galactose 1.10      0.56       0.44    Manose    3.45      1.64       1.41    Klason    25.22     34.35      32.00    Lignin    Acid Sol. 2.74      1.89       0.85    Lignin    Ash       1.35      0.36       0.48    ______________________________________

    ______________________________________               Grams               Feed  Cold Flushed                                 Hot Flushed    ______________________________________    Glucose Equivalents                 2.48    2.00        1.96    Xylose Equivalents                 0.93    0.06        0.04    Arabinose Equivalents                 0.10    0.02        0.02    Galactose Equivalents                 0.06    0.02        0.01    Manose Equivalents                 0.18    0.05        0.04    Klason Lignin                 1.29    1.05        0.06    Acid Sol. Lignin                 0.14    0.06        0.02    ______________________________________     Note: Glucose equivalents = glucose and glucose oligomers expressed in     terms of the weight of total glucose units, etc.

    ______________________________________                 Percent Removed From Feed                 Cold Flushed                          Hot Flushed    ______________________________________    Glucose        19.47      20.96    Xylose         93.28      95.52    Arabinose      76.70      81.33    Galactose      69.43      77.60    Manose         71.45      77.11    Klason Iignin  18.21      28.94    Acid Sol. Lignin                   58.58      82.63    ______________________________________

As one can easily see, the hot flush removed more of each of the sugarsderived from hemicellulose and more of the lignin. The amount of glucoseremoved was clearly not as significant. Most important is the percentageof Klason lignin removed. Previous attempts at acid hydrolysis have notbeen successful at separating appreciable amounts of lignin from thelignocellulose.

By removing greater amounts of hemicellulose and especially greateramounts of lignin, the cellulose remaining is more accessible and morereadily saccharified by cellulase enzyme complex. This has beendemonstrated by using the cellulase digestion process described below inExample 6. The results are as follows:

    ______________________________________    Digestion Percent Digested    Time (hrs)              Cold Flush  Hot Flush Untreated    ______________________________________    0         0           0         0    4         25.6        32.7      4.4    8         42.4        49.4      5.8    24        78.9        82.9      9.2    72        97.2        103.0     11.8    144       98.1        103.8     12.8    ______________________________________

The data appears to indicate that both the rate of cellulose digestionas well as the overall amount of cellulose digested is increased byusing the hot flush technique as compared to the cold flush technique.Therefore, even a single flushing of the reactor while it is undergoingor at the end of prehydrolysis has a beneficial effect to prepare acellulosic product which is more easily degraded by cellulase.

EXAMPLE 9

Using the percolation reactor of the previous Examples, the process wasrepeated with the hybrid poplar lignocellulosic substrate with only hotwater being used as the degrading chemical. The prehydrolyzate liquorwas continuously withdrawn and its components were measured as in theprevious Examples. The solid residue was subjected to the same cellulasetreatment.

A number of different combinations of temperature and time were used torepresent different degrees of severity. These were 30 minutes at 120°C., 30 minutes at 140° C., 30 minutes at 160° C., 60 minutes at 160° C.and 90 minutes at 160° C. Generally speaking, the more severe theconditions, the greater glucan, xylan and klason lignin removal from thesolids and the greater the digestibility.

The substrate and prehydrolyzed solids were analyzed to determine thepercent of each component removed from the solids by using the hot watertreatment. The results are as follows:

    ______________________________________    Component Removal From Lignocellulosic Substrate    Prehydrolysis                Xylan     Klason Lignin                                       Glucan    Conditions  Removed   Removed      Removed    ______________________________________    30 minutes 120° C.                 1%        6%          0.5%    30 minutes 140° C.                 1%       18%          1%    30 minutes 160° C.                20%       17%          1%    60 minutes 160° C.                57%       30%          3%    90 minutes 160° C.                69%       34%          4%    ______________________________________

Levels of furfural and hydroxymethyl furfural were measured and found tobe undetectable in all prehydrolysis runs. The solids were enzymaticallydigested with cellulase. This data is presented as FIG. 5.

While some improvement over no prehydrolysis was noted, thedigestibility of hot water prehydrolyzed lignocellulosic material wasconsiderably less than that noted in FIG. 4 generated from the acidprehydrolysis of the Example above.

The foregoing description of the specific embodiments reveal the generalnature of the invention so that others can, by applying currentknowledge, readily modify and/or adapt for various applications suchspecific embodiments without departing from the generic concept, and,therefore, such adaptations and modifications should and are intended tobe comprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation.

All references mentioned in this application are incorporated byreference.

What is claimed is:
 1. A process for producing sugar fromlignocellulosic material comprising the following steps carried out inthe order given;(a) placing lignocellulosic material in a prehydrolysisflow-through reactor, (b) prehydrolyzing said lignocellulosic material,(c) passing an acidic liquid into the reactor and through saidlignocellulosic material and (d) withdrawing liquid out of saidprehydrolysis flow-through reactor to form a prehydrolyzate liquor, (e)recovering said prehydrolyzate liquor containing oligomers of a pentose,(f) hydrolyzing said oligomers of a pentose, (g) recovering the pentose(h) removing solid material form said flow-through prehydrolysisreactor, (i) adding at least one enzyme capable of digesting cellulosecontained in the lignocellulosic material into glucose to said solidmaterial, and (j) digesting the cellulose in said solid lignocellulosicmaterial with said enzyme to produce glucose, wherein said passing anacidic liquid occurs under prehydrolysis conditions.
 2. A processaccording to claim 1, further comprising adjusting the pH of saidprehydrolyzate liquor after the formation of the prehydrolyzate liquorof step (d).
 3. A process for producing an organic compound selectedfrom the group consisting of: alcohol, ketone, and carboxylic acid fromlignocellulosic material according to claim 1 further comprising thefollowing steps carried out in the order given,adjusting the pH of saidprehydrolyzate liquor, fermenting said pentose in said prehydrolyzateliquor to produce the organic compound, and fermenting said glucose toproduce the organic compound, wherein said passing an acidic liquidoccurs under prehydrolysis conditions.
 4. A process according to claim 3wherein said organic compound is ethanol.
 5. A process for producingsugar from lignocellulosic material comprising;placing lignocellulosicmaterial in a prehydrolysis flow-through reactor, prehydrolyzing saidlignocellulosic material, passing carbon dioxide or carbonic acid intothe reactor and through said lignocellulosic material and removingliquid from said prehydrolysis flow-through reactor to form aprehydrolyzate liquor, lowering the pressure of the prehydrolyzateliquor so that at least some of the carbonic acid is removed fromsolution by volatilization of carbon dioxide, recovering saidprehydrolyzate liquor containing oligomers of a pentose, hydrolyzingsaid oligomers of a pentose contained in the prehydrolyzate liquor,recovering the pentose, removing solid material from said flow-throughprehydrolysis reactor, adding at least one enzyme capable of digestingcellulose into glucose to said solid material, and digesting cellulosein said solid material with said enzyme to produce glucose, wherein saidpassing carbon dioxide or carbonic acid occurs under prehydrolysisconditions.
 6. A process for producing an organic compound selected fromthe group consisting of: alcohol, ketone, and carboxylic acid fromlignocellulosic material according to claim 5 further comprising thefollowing steps after recovering the pentose,fermenting said pentosecontained in said prehydrolyzate liquor to produce the organic compound,fermenting said glucose to produce the organic compound, wherein saidpassing carbon dioxide or carbonic acid occurs under prehydrolysisconditions.
 7. A process according to claim 6 wherein said organiccompound is alcohol.